Drug development is increasingly focusing on targeted therapies directed against membrane receptors. Disruption of signal transduction pathways through pharmacological targeting of relevant membrane receptors has become an effective therapeutic option to is treat e.g. various types of tumors.
Biological molecules, such as monoclonal antibodies (MAbs) as well as small chemical compounds directed against various membrane receptors and other cell proteins on the surface of tumor cells are known to be suitable for anti-tumor therapy for more than twenty years. Mabs specifically bind to their target structures on tumor cells and in most cases also on normal tissues and can cause different effects that dependent on their epitope specificity and/or functional characteristics of the particular antigen. MAbs which bind to an epitope outside the ligand-binding site of membrane receptors (e.g. growth factor receptors with kinase activity) would be expected to induce primarily immune effector functions against the target cell (antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)).
MAbs which bind to an epitope within the ligand-binding site or in its direct neighborhood compete for binding of natural ligands to their receptor and thus reduce or completely inhibit ligand binding and can displace already bound ligands from their receptors. This receptor blockade inhibits ligand-dependent receptor activation and downstream signaling.
Membrane tyrosine kinase receptors in tumor cells are a particularly attractive target in anti-tumor therapies. One receptor type tyrosine kinase subfamily, designated as HER or ErbB subfamily, is comprised of EGFR (ErbB1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Ligands of this subfamily of receptors include epithelial growth factor (EGF), TGF-a, amphiregulin, HB-EGF, betacellulin, heregulin and neuregulins. Among this subfamily the EGFR emerge as one of the most promising targets in anti-tumor therapies.
EGFR is a 170 kD membrane-spanning glycoprotein containing (1) an amino-terminal extracellular domain comprised of 621 amino acid residues, which includes the ligand-binding domain; (2) a single 23-amino-acid transmembrane-anchoring region which may contribute to stability; and (3) a 542-amino-acid carboxyl-terminal intracellular domain which possesses tyrosine kinase activity that activates cytoplasmic targets. Examples of ligands that stimulate EGFR include epidermal growth factor (EGF), transforming growth factor-cc (TGF-a), heparin-binding growth factor (HBGF), (3-cellulin, and Cripto-1. Binding of specific ligands results in EGFR autophosphorylation, activation of the receptor's cytoplasmic tyrosine kinase domain and initiation of multiple signal transduction pathways that regulate tumor growth and survival.
It should be remarked that receptor protein tyrosine kinases, such as EGFR kinase are able to undergo both homo- and heterodimerization, wherein homodimeric receptor combinations are less mitogenic and transforming (no or weak initiation of signaling) than the corresponding heterodimeric combinations (Yarden and Sliwkowski, 2001, Nature Reviews, Molecular cell Biology, volume 2, 127-137; Tzahar and Yarden, 1998, BBA 1377, M25-M37).
Oncogenic transformation due to aberrant EGFR signaling can be a consequence of several different mechanisms, including receptor overexpression.
The epidermal growth factor receptor (EGFR) is aberrantly activated in a variety of epithelial cancers and has been the focus of much interest as a therapeutic target in anti-tumor therapy. EGFR is involved in critical cellular processes such as proliferation, differentiation and apoptosis (Hubbard and Miller, 2007). Misregulation of EGFR, through overexpression or mutation, leads to constitutive activity or impaired receptor downregulation and can cause malignant transformation of the cell (Mendelsohn and Baselga, 2006).
One of the most important strategies to pharmacologically target EGFR, includes monoclonal antibodies (MAbs) which compete with activating EGFR ligands for binding to the ligand-binding side in the extracellular receptor domain.
The first strategy used clinically to target aberrant EGFR signaling in malignant cells was the use of MAbs. Anti-EGFR antibodies not only disrupt receptor/ligand interactions, blocking aberrant signaling and thus tumor cell proliferation and growth, but they may also modulate anti-tumor effectors via antibody-dependent cellular cyto-toxicity (ADCC). Natural killer (NK) cells mediate ADCC by recognizing the carboxyl-terminal ends of antibody molecules via the low-affinity receptor for IgG, FcyRIIIA/CD16. NK cells therefore can closely interact with antibody-coated tumor cells and destroy cells via necrosis and apoptosis.
The first murine anti-EGFR MAb developed showed good anti-tumor activity in animal models. However, their clinical use was limited due to the high incidence of human antimurine antibodies in patients, resulting in reduced efficacy. In response to this disadvantage, researchers developed chimeric and humanized forms of anti-EGFR MAbs.
Cetuximab (IMC-C225, Erbitux©) a chimeric anti-EGFR antibody, was the first Mab of this type that successfully completed clinical trials and was launched in 2003 as a treatment for several cancers. Cetuximab is described e.g. in WO96/40210.
There are a number of other anti-EGFR antibodies under active clinical development for the treatment of cancer. One of them is matuzumab.
Matuzumab (EMD-72000) is a humanized IgG1 MAb that binds with high specificity and affinity to EGFR. Matuzumab is described in WO1992/015683. It has been shown in animal tumor xenograft models to have potent inhibitory activity against human cancers, including head and neck, gastric, pancreatic and lung cancers. Matuzumab was shown to block EGF binding to EGFR, thereby inhibiting downstream signaling pathways, and it may also act via ADCC through FcR binding on immune cells. Matuzumab was selected for further development as a treatment for cancer. Matuzumab exhibited antitumor activity against surgical specimens of EGFR expressing human lung (LXFA629) and gastric (GXF251) adenocarcinomas and pancreas adenosquamous carcinoma (PAXF546) that were insensitive to chemotherapeutic drugs (bleomycin, cisplatin, vindesine, paclitaxei, ifosfamide) and implanted s.c. in nude mice. Treatment with matuzumab (0.5 or 0.5 mg/mouse i.p. once weekly for 2 weeks starting when tumors reached 70-120 mm3) was well tolerated and effective against all 3 tumor types. Complete remissions were observed in 83% and 87%, respectively, of animals bearing gastric and lung carcinomas treated with the higher dose. Marked reductions in pancreatic tumors were observed, such that a mean tumor volume of 31% compared to controls was obtained (27). The anti-tumor efficacy of matuzumab (40 mg/kg biweekly) in mice bearing orthotopic human L3.6pI pancreatic tumors was shown to be enhanced by simultaneous treatment with gemcitabine (100 mg/kg biweekly). Treatment with either agent alone caused a reduction in tumor size and lymph node and liver metastases. These effects were markedly enhanced by combination treatment. Treatment with matuzumab alone or in combination with gemcitabine also significantly decreased microvessel density and proliferative indices. Results from in vitro and in vivo studies further suggest that the anti-tumor effects of matuzumab involve ADCC.
A combination of cetuximab and matuzumab results in a synergistic effect of tumor regression (WO2004/032960).
A combination of anti-EGFR antibodies with chemotherapeutic agents elicits also an enhanced anti-tumor effect (e.g. EP0667165).
Besides Mabs there are numerous small chemical drugs which are known to be potent inhibitors of membrane receptors. Regarding ErbB receptors they block the binding site of the natural ligands, or block the tyrosine residues of the binding site of the receptor kinase, thus preventing phosphorylation and further cascade signaling. One representative showing high efficacy in clinical trials is Iressa™ (ZD-1839) which can be applied for NSCLC indication (non-small cell lung cancer).
In contrast to these conventional cytotoxic drugs, targeted therapies cannot be applied at the maximum tolerated dose (MTD) since it may interact with other signal pathways if administered in supra saturating doses. In oncology or hematology targeted therapies are combined with these cytotoxic drugs frequently. The combination therapies of targeted drugs may influence safety profile. This problem leads to the need for a dose reduction of the MAbs. Thus, a target effective dose (TED) has to be defined to ensure a sufficient MAb dosing. The assessment of receptor binding saturation significantly contributes to the definition of a dose rationale i.e. the TED and is superior to investigate effectiveness on downstream signaling that may be altered by salvage pathways.